Although critical periods for structural and many behavioral anomalies due to prenatal alcohol exposure have been identified, little is known about the effects of ethanol (EtOH) on preimplantation embryos. Preliminary studies from our laboratory indicate that EtOH exposure during preimplantation can cause implantation to occur earlier than normal, possibly resulting in a disruption of maternal-fetal synchrony. In our proposed project, an in vitro culture system for mouse pre- and peri-implantation embryos will be used to assess the direct effects of EtOH exposure during early pregnancy independent of maternal confounders. Based on our preliminary studies that clearly demonstrate an accelerated rate of embryogenesis among embryos treated with 0.1 to 0.4% (w/v) EtOH, the mechanism underlying this effect will be explored. The effect of this treatment appears to be specific, rather than globally cytotoxic, since treated embryos can develop normally to term. Using this model, we will identify the biochemical events that follow the exposure of cells to EtOH and develop a working model of the physiological effects of EtOH on embryonic cells. Based on our preliminary results, we hypothesize that EtOH accelerates preimplantation development by perturbing the plasma membrane and altering components of the signal-transduction pathway, leading to a precocious change in the expression of genes that regulate subsequent development. This hypothesis will be tested using normal and EtOH-treated embryos by examining selected components of the signal transduction pathway and by identifying those genes that undergo altered expression in response to EtOH. Using immunohistochemistry and micro-enzymatic assays, the activity of growth factor receptors will be measured, and where appropriate, the associated protein tyrosine kinase activity, also. Other aspects of the signal transduction pathway that will be examined include phospholipase C activity, protein kinases C activity, intracellular Ca2+ levels, and the expression of the putative transcription factors, c-myc, c-fos, and c-jun. to identify the gene transcripts that are expressed at increased or decreased levels during EtOH-induced acceleration of embryo development, EtOH-regulated genes will be identified by the technique of subtraction hybridization with normalized cDNA libraries prepared using mRNA isolated from control and ETOH-treated embryos. This technology has the capacity to select treatment-specific cDNAs that are representative of all abundance classes of mRNA in a form amenable to identification and characterization. The unique transcripts will be sequenced and, where possible, identified using databases. In this manner, a catalogue of both known and unidentified, unique sequences modulated by EtOH will be acquired. This resource will be available to us and others to subsequently determine the role of these unique gene products in EtOH perturbation of preimplantation and fetal development.
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